DESCRIPTION (Abstract of the application) The development of gene therapies for chronic diseases critically depends on the development of non-toxic vectors that are capable of achieving long term gene expression. All currently used vectors have a limited' capacity for sustained gene expression. In the relatively immune-privileged central nervous system (CNS) it is possible to study vector transcriptional silencing, one of the major mechanisms limiting prolonged gene expression. Our hypothesis is that the extent and duration of expression of a somatically transferred transcription unit is a function of chromatin organization. We propose that transcriptional silencing is a consequence of heterochromatin formation on the vector genome. We will directly test this hypothesis by analyzing gene expression and chromatin structure in the HSV amplicon vector. In addition, we will examine one of the predictions of the hypothesis: inclusion within amplicon vectors of cis elements will direct chromatin, organization and increase the likelihood for long term gene expression. Specifically, we will test whether a matrix attachment region (MAR) element placed in a HSV amplicon vector imparts resistance to heterochromatin formation and its consequence- transcriptional silencing. A glucocorticoid-responsive transcription unit will be used because it has a DNA-protein signature recognizable in biochemical studies to examine chromatin structure I on vector genomic DNA. This project focuses on HSV amplicon vectors, versatile plasmid-based vectors that do not contain any viral genes and which can be prepared free of helper virus. Achieving long term expression from HSV amplicon vectors requires an understanding of how to direct the organization of the transcription unit within the target cell. We believe that the characterization of chromatin structure as it pertains to expression from the HSV amplicon vector in the CNS will have implications for extending gene expression from other episomal vectors and in different tissues.